IKKb-Mediated Resistance to Skin Cancer Development Is ......Oncogenes and Tumor Suppressors...

Oncogenes and Tumor Suppressors

IKKb-Mediated Resistance to Skin CancerDevelopment Is Ink4a/Arf-DependentAngustias Page1,2,3, Ana Bravo4, Cristian Suarez-Cabrera1,2,3,Josefa P. Alameda1,2,3, M. Llanos Casanova1,2,3, Corina Lorz1,2,3,Carmen Segrelles1,2,3, Jos�e C. Segovia5,6, Jes�us M. Paramio1,2,3,Manuel Navarro1,2,3, and Angel Ramirez1,2,3

Abstract

IKKb (encoded by IKBKB) is a protein kinase that regulatesthe activity of numerous proteins important in several signalingpathways, such as the NF-kB pathway. IKKb exerts a protumori-genic role in several animal models of lung, hepatic, intestinal,and oral cancer. In addition, genomic and proteomic studies ofhuman tumors also indicate that IKBKB gene is amplified oroverexpressed in multiple tumor types. Here, the relevance ofIKKb in skin cancer was determined by performing carcinogen-esis studies in animal models overexpressing IKKb in the basalskin layer. IKKb overexpression resulted in a striking resistanceto skin cancer development and an increased expression ofseveral tumor suppressor proteins, such as p53, p16, and p19.Mechanistically, this skin tumor–protective role of IKKb is

independent of p53, but dependent on the activity of theInk4a/Arf locus. Interestingly, in the absence of p16 and p19,IKKb-increased expression favors the appearance of cutaneousspindle cell–like squamous cell carcinomas, which are highlyaggressive tumors. These results reveal that IKKb activity pre-vents skin tumor development, and shed light on the complexnature of IKKb effects on cancer progression, as IKKb can bothpromote and prevent carcinogenesis depending on the cell typeor molecular context.

Implications: The ability of IKKb to promote or prevent carcino-genesis suggests the need for further evaluation when targetingthis protein. Mol Cancer Res; 15(9); 1255–64. �2017 AACR.

IntroductionCancer is a multifactorial disease, caused by alterations in

oncogenes that activate growth-promoting signaling cascadesor in tumor suppressor genes whose inactivating mutationslead to DNA reparation defects, genomic instability, or defec-tive control of cell proliferation. Multiple studies indicate thatderegulation of the activity of NF-kB can result in cancerdevelopment. NF-kB is a conserved family of ubiquitous tran-scription factors that regulates the expression of many genes

involved in cell proliferation, survival, apoptosis, and otheressential processes, which is usually overactivated in inflam-matory diseases and in cancer (reviewed in ref. 1). NF-kB isusually bound to inhibitory proteins of the I-kB family, whichupon activation are phosphorylated by the IKK complex anddegraded by the proteasome; consequently, NF-kB is releasedand modifies the expression of a myriad of genes containingspecific DNA-regulatory elements.

The multiprotein complex IKK is formed by the regulatorysubunit IKKg and the catalytic subunits IKKa and IKKb. Thefirst known function of IKKb kinase was the regulation ofNF-kB activation upon proinflammatory stimuli, but it alsoperforms important NF-kB–independent functions, as IKKbphosphorylates a plethora of target proteins, thus controllingtheir activity and stability. Important tumor suppressor genes(TSG), like p53 (2) and p16 (3), are targets of IKKb; the sameholds true for other relevant proteins in cancer (as MDM2, p63family members, ATM, IRS-1, TSC-1, or FOXO3a; reviewed inrefs. 4–6). Furthermore, studies performed in MCF-7 mamma-ry gland cells revealed the astonishing variety of IKKb sub-strates, as more than 4,000 proteins containing phosphoryla-tion sites for IKKb were found (7). In this way, IKKb affectsmTOR, insulin, and Wnt signaling, immune responses, autop-hagy, response to DNA damage, and cell transformation. Insummary, IKKb acts over many different cellular processes,including tumoral transformation, both dependently and inde-pendently of NF-kB.

Animal models with altered expression of members of theNF-kB signaling pathway highlight the importance of theseproteins in skin cancer. For example, mice lacking p65, the

1Molecular Oncology Unit, Centro de Investigaciones Energ�eticas, Medioam-bientales y Tecnol�ogicas (CIEMAT), Madrid, Spain. 2Cell andMolecular OncologyGroup, Institute of Biomedical Research, Universitary Hospital 12 de Octubre,Madrid, Spain. 3Centro de Investigaci�on Biom�edica en Red de C�ancer (CIBER-ONC), Spain. 4Department of Anatomy, Animal Production and VeterinaryClinical Sciences, Faculty of Veterinary Medicine, University of Santiago deCompostela, Lugo, Spain. 5Hematopoietic Innovative Therapies Division. Centrode Investigaciones Energ�eticas, Medioambientales y Tecnol�ogicas (CIEMAT),Centro de Investigaciones Biom�edicas en Red de Enfermedades Raras(CIBERER), Spain. 6Advanced Therapies Mixed Unit, Instituto de Investigaci�onSanitaria-Fundaci�on Jim�enez Díaz (IIS-FJD), Madrid, Spain.

Note: Supplementary data for this article are available at Molecular CancerResearch Online (http://mcr.aacrjournals.org/).

Corresponding Author: Angel Ramirez, Centro de Investigaciones Energ�eticas,Medioambientales y Tecnol�ogicas (CIEMAT), ED 70, Avda. Complutense 40,Madrid 28040, Spain. Phone: 34-913460882; Fax: 34-913466484; E-mail:[email protected]

doi: 10.1158/1541-7786.MCR-17-0157

�2017 American Association for Cancer Research.

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main NF-kB subunit, are resistant to skin carcinogenesis (8);the expression in proliferative skin cells of a super repressorform of I-kBa leads to hyperplasia, to a strong inflammatoryresponse and finally to the development of squamous cellcarcinomas (SCC; ref. 9); IKKa also has a role in skin cancer,showing protumoral or tumor-suppressive activity dependingon the experimental setting (10, 11).

Regarding the relationship between IKKb and cancer, a tumor-promoting activity of IKKb in intestinal, hepatic, lung, and pan-creatic cancer has been reported (12–17). In myeloid cells, bycontrast, IKKb is implicated in orchestrating an antitumoralimmune response against melanoma cells (18). So, althoughIKKb promotes tumor development in general, its actual effectover tumor growth and malignancy is cell type specific. We havepreviously described that IKKb promotes tumor development inoral epithelia (19), but its role in skin cancer has not beendetermined yet. In this work, we explore the effect of IKKb inskin cancer and the underlying molecular pathways by usingtransgenic mice with increased levels of epidermal IKKb andabsence of p53 or p16/p19 tumor suppressor proteins. At differ-ence to the generally reported protumoral role of IKKb in epi-thelial carcinogenesis, IKKb showed a strong tumor suppressoractivity in skin tumorigenesis that is independent of p53; inter-estingly, it is mediated by the proteins coded by the Ink4a/Arflocus (p16 and/or p19). In the absence of p16 and p19, we foundthat IKKb promotes the emergence of spindle SCCs, a rare variantof malignant skin tumor. These results have implications for theimplementation of antitumoral or anti-inflammatory therapies inskin, as IKKb is considered a potential target for the developmentof these types of interventions.

Materials and MethodsMice and treatments

Mouse experimental procedures were performed according toEuropean and Spanish regulations and were approved by theEthics Committee for AnimalWelfare of CIEMAT and by the legalauthority (protocol codes BME02/10 and PROEX086/15). Trans-genic mice used in this work have been described previously: lineL1of K5-IKKbmice in B6D2 hybrid background (20); TgAC (FVB/NTac-Tg(Hba-x-v-Ha-ras)TG.ACLed) mice (21), p53EKO mice(22, 23), and Ink4a/ArfKO mice (lacking p16 and p19 proteinsin FVB background; ref. 24). K5-IKKb mice are available atEuropean Mouse Mutant Archive (code EM: 09179).

The mice used in this study were viable and lack any evidentalteration. In the breedings, all the genotypeswere obtained in theexpected ratios.

For two-stage skin carcinogenesis experiments, back skinsof mice were initiated two days after shaving by treatmentwith 100 mg of 7,12-Dimethylbenz[a]anthracene (DMBA,Sigma-Aldrich), and promoted with 5 mg of 12-O-tetradeca-noylphorbol-13-acetate (TPA, Sigma-Aldrich) in 200 mL ofacetone twice weekly during 12 weeks. In carcinogenesis experi-ments in TgAC background, skins were TPA-treated twiceweekly for 12 weeks. Tumor number and size were recordedweekly. For bromodeoxyuridine (BrdUrd) labeling, mice wereinjected intraperitoneally with BrdUrd (0.1 mg/g weight, Sig-ma-Aldrich); 1.5 hours later, mice were sacrificed and the skinwas harvested. For hyperplasia induction, mice back skin wasshaved and topically treated twice with 5 mg of TPA or vehicle atdays 3 and 5 after shaving.

Genotyping of miceGenotyping of K5-IKKb and of p53 and Ink4a/Arf loci were

performed by PCR analysis of tail DNA, as described previously(20, 23, 25).

Histology and IHCMouse tissues were dissected and fixed in 10% buffered for-

malin or 70% ethanol and embedded in paraffin. Five-micronthick sections were used for hematoxylin and eosin (H&E) stain-ing or IHC preparations. Antibody references are provided inSupplementary Material and Methods. Immunoreactivity wasrevealed using an ABC avidin–biotin–peroxidase system and ABCsubstrate (Vector Laboratories), and counterstained slightly withhematoxylin.

Quantification of BrdUrd staining and vessel densityFor quantification of BrdUrd staining in Fig. 1E, the number of

BrdUrd-positive cells in 600 to 800 basal cells in four epidermalhyperplasias from 4 TgAC/K5-IKKb mice and in 6 papillomasfrom 4 TgAC mice were counted. For vessel density (Fig. 1F), thenumber of blood vessels were counted in 10 different fields (20�objective) in 5 different TgACmice and 4 different TgAC/K5-IKKbmice topically treated with TPA for 3 weeks.

Determination of epidermal CD34þ cellsFor the flow cytometry analysis shown in Supplementary Fig.

S2B and S2C,mean values were obtained from the analysis of fiveindividual K5-IKKb and wild-type (wt) mice. Similar results wereobtained in three independent experiments. The images shown inSupplementary Fig. S2D and S2E are representative of the dataobtained in the analysis of 3 wt and 3 K5-IKKb 10-week-oldmice.Data in Supplementary Fig. S2F–S2H comes from the analysis of 4animals of each genotype.

Protein extraction and Western blot analysisWhole-cell protein extracts from mouse tumors and tissues

were prepared using standard techniques. Protein content wasdetermined by the Bradford colorimetric protein assay (Bio-RadLaboratories). Antibody references are provided in Supplemen-tary Material and Methods.

Keratinocyte culturePrimary keratinocytes were obtained from skins of 2-day-old

mice and cultured as describedpreviously (20). Plateswith 3-day–cultured keratinocytes were scrapped-off, pelleted, and the pro-tein extracts subjected to Western blotting.

Statistical analysisData are expressed asmean� SD. Statistical significance of data

was assessed using the unpaired, two-tailed Student t test or Fisherexact test. P values <0.05 were considered significant.

ResultsIKKb activity protects against skin cancer

With the aim of determining the role of IKKb in nonmelanomaskin cancer (NMSC), we performed DMBA-TPA carcinogenesisassays in transgenicmice overexpressing IKKb in basal cells of skin(K5-IKKbmice) and in nontransgenic littermates (n¼ 17 for eachgenotype). K5-IKKb mice developed fewer tumors than wt mice(Fig. 1A). In addition, fewer K5-IKKbmice generated tumors, and

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Mol Cancer Res; 15(9) September 2017 Molecular Cancer Research1256




those tumors were smaller than wt tumors (not shown); all thesedata suggest an antitumoral activity of IKKb in skin. Given the lowtumor number obtained in this carcinogenesis protocol, we

aimed to confirm these data in other genetic backgrounds moreprone to skin tumorigenesis. We used TgACmice, which express amutatedHa-Ras oncogene in skin (26).We generated cohorts of 9

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Protective function of IKKb in mouse skin tumorigenesis. A, Tumor multiplicity in K5-IKKb transgenic mice and wt littermates in C57BL6/J � DBA2/J hybridbackground. The lownumber of tumors permouse is due to the low sensitivity to skin carcinogenesis protocols of themice in this background.B, Tumormultiplicity inK5-IKKb transgenic mice and control littermates in TgAC background. Tumor burden is much more elevated than in Fig. 1A by the increased sensitivity ofTgAC mice to tumor development. C, Examples of the macroscopic appearance of TgAC and TgAC/K5-IKKb mice subjected to TPA treatment at week 12 andexamples of the H&E staining of one of the lesions obtained. TgAC mice developed numerous exophytic papillomas generally greater than 4 mm in diameter. Incontrast, TgAC/K5-IKKb developed a lower number of smaller tumoral lesions. Note that the TgAC/K5-IKKbmouse shown in the macroscopic image is the one withthe highest number of tumoral lesions in this genotype. D, Representative images of the IHC analysis of IKKb expression, proliferation (measured as BrdUrdincorporation), and blood vessels supply (Smaexpression), in tumoral lesions of the indicated genotypes. E,Quantification of BrdUrd staining. F,Quantification of thenumber of blood vessels in the dermis of TPA-treated skins of the indicated genotypes. � , P < 0.05; �� , P < 0.01. Scale bars, 100 mm.

Antitumoral Activity of IKKb in Skin

www.aacrjournals.org Mol Cancer Res; 15(9) September 2017 1257




mice bearing and 11mice without the K5-IKKb transgene in TgACbackground, which were subjected to topical treatment with TPAto induce papilloma formation. Although 100% of the controlTgACmice developed papillomas byweek 7 of treatment, none ofthe TgAC/K5-IKKbmice developed tumors at this time point (P <0.0001), and only aroundhalf of themdeveloped tumors byweek12 (not shown). Furthermore, the number of papillomas permouse and their size was markedly diminished in TgAC/K5-IKKbmice compared with TgAC mice (Fig. 1B and C). There weremarked macroscopic and microscopic differences between bothgenotypes: while TgAC lesions were truly exophytic papillomastypical of this carcinogenesis protocol, the lesions obtained inTgAC/K5-IKKb were much smaller papillomatous epidermalhyperplasias (Fig. 1C), which proliferated less and induced aweaker angiogenic response than TgAC papillomas (Fig. 1D andE). This weaker angiogenic response was also observed in TgAC/K5-IKKb skin TPA-treated for 3 weeks (before the development ofskin lesions) when compared with TgAC skin (Fig. 1F). Theseresults indicate that IKKb precludes skin tumor formation. Trans-genic mice express IKKb in a variety of tissues; the markeddifference in IKKb effect over tumorigenesis between skin andoral epithelia do not seem to be related with differences in theexpression level of IKKb between both tissues (19).

K5-IKKb skin shows minor differences in immunecell populations

Inflammation by infiltrating immune cells facilitates the acqui-sition of the fundamental features of cancer (27) and deeplyaffects skin tumor development (28). One important function ofIKKb is the regulation of the inflammatory process by controllingNF-kB activity. So, the antitumoral activity of IKKb in skin couldbe mediated by differences in skin resident immune cell popula-tions. We studied by flow cytometry the number of differentimmune cells both in epidermis and in dermis of wt mice andK5-IKKb littermates (Supplementary Fig. S1A and S1B). Interest-ingly, the percentage of CD45þ cells is roughly double in dermisthan in epidermis of normal mice (9.3% vs. 4.3%); these cells aremainly T lymphocytes (CD3þ cells), both inflammatory (CD3þ,Tbþ) and skin-resident T cells (CD3þ, Tgdþ). We also detectedother immune cell populations potentially relevant in carcino-genesis, as macrophages (CD11bþ), dendritic cells (CD11cþ),and natural killer cells (Supplementary Fig. S1A). In K5-IKKbepidermis, we observed a modest, nonsignificant decrease inCD3þ cells (both in Tbþ and in Tgdþ subpopulations), a slight

increase in macrophages, and a more prominent diminution inNK cells (Supplementary Fig. S1B). In dermis, all the populationsstudiedwere roughly equally abundant in Tg and inwtmice. Fromthese results, we conclude that the minor changes observed in thefrequency of immune cells (T lymphocytes and myeloid cells) donot seem responsible for the marked differences in skin cancersusceptibility between K5-IKKb Tg and wt mice.

Characterization of the stem cell population in skinof K5-IKKb transgenic mice

The decreased susceptibility to tumoral transformation inK5-IKKb mice could be related to differences in the amount ofcells able to originate a tumor. CD34 is a cell surface markerexpressed by skin stem cells in the bulge region of the hairfollicles (29), which is needed for papilloma formation inmice (30). CD34þ cells become more abundant during theprocess of malignization (31), and CD34þ cells isolated fromprimary tumors can originate new tumors if transplanted tosecondary recipients (32). Therefore, CD34 is considered amarker of stem cells and of cancer stem cells in skin. To assesswhether the lower frequency of skin cancer in IKKb-overexpres-sing mice is related to a reduction in the number of skin CD34þ

cells, we studied the abundance and cell-cycle distribution ofthese cells (Supplementary Fig. S2A–S2C). Surprisingly, K5-IKKb mice have roughly double the number of CD34þ cellsthan their nontransgenic counterparts (Supplementary Fig. S2Aand S2B). As expected, these CD34þ cells were located in thehair follicle bulge both in wt mice and in K5-IKKb mice skin(Supplementary Fig. S2D and S2E), but they seem to cycleslightly less actively in Tg than in wt mice (statistically non-significant, Supplementary Fig. S2C). In BrdUrd label-retainingassays, we did not detect a reduction in the stem cell populationin K5-IKKb mice (Supplementary Fig. S2F–S2H). Therefore, weconclude that the decreased sensitivity to skin cancer found inK5-IKKb mice is not due to the lack of stem cells able togenerate tumors, or to differences in their location or prolifer-ative state.

K5-IKKb keratinocytes express increased levelsof tumor suppressor genes

To understand the differences observed in tumoral predispo-sition between wt and K5-IKKb transgenic skin, we studied theexpression of several TSGs important in tumoral transformation.We analyzed p53 (encoded by the TSG most frequently mutated

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Increased expression of p53, p19, andp16 in K5-IKKb epidermalkeratinocytes. A, Western blotanalysis of cultured skin keratinocytes.B and C, IHC staining of p19 inhyperplastic back skin sections of theindicated genotypes; note theincreased staining of basal skincells of K5-IKKb transgenic micecompared with wild-type mice(arrowheads). D, qRT-PCR analysis ofthe expression of several tumorsuppressor proteins in RNA samplesfrom skin keratinocytes of transgenicK5-IKKb and wild-type mice.

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in cancer), p19 and p16 proteins (products of the transcription ofthe Ink4a/Arf locus, implicated in the functionality of p53 and Rbproteins, respectively), and p21 (a protein transcriptionally reg-ulated by p53 that controls cell-cycle progression). As the analysisof these proteins in complete skin by Western blot analysis is notappropriate, due to the variety of cellular types present in skin, westudied cultured skin keratinocytes from K5-IKKb and wt mice.We consistently found that transgenic keratinocytes express

increased amounts of p53, p16, and p19 (Fig. 2A). We confirmedthe result for p19by IHCstaining in TPA-treated skin sections (Fig.2B and C: 62% of basal nuclei were intensely stained in Tg skin, atdifference with theweak staining found in 24%of the nuclei in wtmice). In accordance with the data obtained in the Western blotanalysis, we found, in qRT-PCR analysis of RNAs from Tg- andwt-cultured skin keratinocytes, a significant increase for p16, p19,and p53 in Tg keratinocytes, but not for p21 (Fig. 2D). Altogether,

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p53 is not needed for the IKKb tumor-protective activity.A, Tumor multiplicity in K5-IKKb transgenic mice andcontrol littermates in p53EKO background subjected to atwo-stage DMBA/TPA carcinogenesis protocol(n ¼ 7 in each group). Differences between bothgenotypes from week 8 to week 15 are statisticallysignificant (P <0.01; Student t test).B, Size distribution ofskin tumors along the experiment. Note the reducedpercentage of large- and medium-sized tumors inp53EKO/K5-IKKb mice in comparison with p53EKO mice.C, Histopathologic classification of tumors obtained inp53EKO and p53EKO/K5-IKKb mice after treatment withDMBA and TPA. The percentages of malignant tumorsare indicated (P < 0.001, Fisher exact test).D–G, Histologic aspect of benign and malignant tumorsof the indicated genotypes;D andE are benign squamouspapillomas and F and G are undifferentiatedSCCs. H, Western blot analysis ofimportant proteins for tumor progression in3 SCCs arisen in mice of each of the indicated genotypes.In the lane marked sk, an extract from K5-IKKbcultured skin keratinocytes was loaded. Note increasedp21, p19, and p16 levels in p53EKO/K5-IKKb carcinomas.Scale bars, 100 mm.






these results suggest that the skin tumor–protective activity ofIKKb could well be mediated by the increased amount of p16,p19, and p53 in skin keratinocytes.

The skin tumor–suppressive activity of IKKb isindependent of p53

We aimed to analyze the implication of p53 in the skin tumor–suppressive activity of IKKb. To this end, we crossed K5-IKKbmicewith p53fl/fl/K14-Cre mice (22, 33). These mice efficiently deletep53 exons 2 to 10 in the vastmajority of epidermal cells (23), andso we named them p53EKO mice; mice carrying both geneticmodifications (i.e., p53EKO/K5-IKKb mice), simultaneously lackp53 and overexpress IKKb in skin basal proliferative cells, whereskin tumors arise. We performed a two-stage skin carcinogenesisexperiment and compared the rate of tumor appearance, tumorsize, and histologic classification between p53EKO/K5-IKKb andcontrol p53EKOmice (Fig. 3). Interestingly, p53EKO/K5-IKKbmicealso showed a great decrease in the mean number of skin tumorsper mice along all the experiment (Fig. 3A), similar to the resultsobtained in p53wt background (Fig. 1A and B). Also, skin tumorsin p53EKO/K5-IKKb mice were smaller than in p53EKO mice,being the proportion of medium and large tumors (diameter of2–5 mm, and >5 mm, respectively) greater in p53EKO mice (Fig.3B). Macroscopically, most tumors arising in p53EKO/K5-IKKbmice were small, exophytic, and papillomatous (although four ofthe animals of this genotype developed only one tumor each offaster growth andapparentlymoremalignant,withulceration andinfiltration of the subcutaneous tissue, not shown). Most p53EKO

mice, in contrast, developed multiple sessile-infiltrating tumors,presumablymalignant. We assessed histologically 51 p53EKO and26 p53EKO/K5-IKKb tumors collected at weeks 13–18 (Fig. 3C).We found several tumor types, and classified them as benigntumors (including squamous papillomas, trichoepitheliomas,adenosquamous tumors, and basosquamous tumors) or malig-nant tumors (including squamous papillomas with invasivemicrocarcinoma foci and more malignant undifferentiatedSCCs). Interestingly, the percentage of malignant tumors waslower in p53EKO/K5-IKKb mice than in p53EKO mice (Fig. 3C).Representative examples of these tumors are shown in Fig. 3D–G.Benign tumors keep a clear basal membrane that prevents inva-sion (arrowheads in Fig. 3D and E). Carcinomas were frequentlyaccompanied by inflammatory cells, and showed highly dysplas-tic keratinocytes invading underlying dermis, with condensednuclei (karyopyknosis), variable in size (anisokariosis), probablyas a consequence of p53 absence (asterisks in Fig. 3F and G). Thetendency to develop of lessmalignant tumors in p53EKO/K5-IKKb

mice was further confirmed by the decreased expression of mar-kers associated to malignant transformation, such as keratin K13and P-Akt, even in the few SCCs obtained in this genotype(Supplementary Fig. S3). In summary, these results indicate thatIKKb overexpression leads to the development of fewer andless malignant tumors in mice lacking skin p53. Western blotanalysis of SCCs from both genotypes confirmed that tumorsoriginated from cells lacking p53, as expected (Fig. 3H). In addi-tion, p53EKO/K5-IKKb carcinomas showed increased p19 and p16levels, in accordance with the results shown in Fig. 2A for culturedkeratinocytes, and also of p21. When we studied Stat-3 and Akt,two proliferative pathways frequently activated in carcinogenesis,we did not find marked differences between p53EKO and p53EKO/K5-IKKb tumors. Altogether, these results indicate that the tumor-suppressive activity of IKKb in skin is not mediated by p53.

In addition to the DMBA/TPA carcinogenesis protocol, wealso studied the combined effect of IKKb overexpression andp53 absence over spontaneous tumorigenesis. We generatedadditional cohorts of p53EKO and p53EKO/K5-IKKb mice, fol-lowed tumor appearance over time, and classified histologicallythe tumors generated (Table 1A). Skin tumors were mainlySSCs of different degrees of differentiation. Interestingly, thepercentage of mice with tumors was lower in p53EKO/K5-IKKbthan in p53EKO mice, and the mean number of tumors per micewith tumors was also lower in p53EKO/K5-IKKb mice (1.4 vs.1.8). Furthermore, the percentage of skin tumors was roughlyhalf in mice overexpressing IKKb (38% vs. 78%; P < 0.001).Collectively, these results reinforce, in a different experimentalin vivo situation, the protective effect of IKKb in skin cancer andconfirm its independence from p53.

Ink4a/Arf locus mediates the skin tumor–suppressiveactivity of IKKb

We next wondered whether the tumor-suppressive activity ofIKKb could be mediated by p16 and p19, which are coded by thesame Ink4a/Arf locus. Heterozygousmice for an Ink4a/ArfKO allele(24) were crossed with K5-IKKb mice and backcrossed with FVBmice for three generations; we obtainedmice homozygous for theInk4a/ArfKO allele (lacking p16 and p19 in all their cells), andsimultaneously bearing the K5-IKKb transgene. We performed aDMBA/TPA skin carcinogenesis experiment in 11 Ink4a/ArfKO

mice and 12 Ink4a/ArfKO/K5-IKKb mice. This experiment couldnot be extended for more than 14 weeks after DMBA applicationdue to humanitarian reasons, given the fast growth or the malig-nant aspect of some of the tumors. Interestingly, in the absence ofthe proteins coded by the locus Ink4a/Arf, there were no

Table 1. Histopathologic classification of spontaneous tumors in: p53EKO and p53EKO/K5-IKKb mice (A) and Ink4a/ArfKO and Ink4a/ArfKO/K5-IKKb mice (B)

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Mice analyzed 44 45 28 40Mice with tumors (%) 23 (52%) 17 (38%) 5 (18%) 18 (45%)Total tumor number 41 24 7 23Tumors per mouse 1,8 1,4 1,4 1,3Skin tumorsa 32 (78%) 9 (38%) 2 (29%) 6 (26%)Other tumorsa,b 9 (22%) 15 (62%) 5 (71%) 17 (74%)Average age of skin tumor appearance (months) 10 9,5 8,5 6,5aThe percentage versus total tumor number is shown.bNon-skin tumors obtained in p53EKO background were 4 oral and 5 mammary gland tumors in p53EKO mice, and 9 oral and 6 mammary gland tumors in p53EKO/K5-IKKbmice. Non-skin tumors obtained in Ink4a/ArfKO backgroundwere 5 hematologic tumors in Ink4a/ArfKOmice, and 4 hematologic and 13 oral tumors in Ink4a/ArfKO/K5-IKKb mice.

Page et al.





differences in tumor burden between animals carrying or lackingthe K5-IKKb transgene (Fig. 4A). The histopathologic classifica-tion of the tumors analyzed (121 in total) is shown in Fig. 4B. Ofnote, due to the absence of p16 and p19, and in spite of therelatively short period of time of the experiment, the majority ofthe tumors were SCCs of varying levels of differentiation. Incontrast, K5-IKKb mice bearing two wt copies of the Ink4a/Arflocus resulted in a marked lower amount of SCCs that arose atlater times than SCCs in Ink4a/ArfKO background. In addition, allthe SCCs observed in Ink4a/Arf wt/wt were well differentiated SCCs(Supplementary Fig. S4A–S4C).

Interestingly, in contrast to the results obtained in wt andp53EKO backgrounds (Figs. 1 and 3), malignancy was augmentedin Ink4a/ArfKO/K5-IKKb tumors, as the percentage of undifferen-

tiated SCCs and spindle cell SCCs (SpSCCs) was much higher inInk4a/ArfKO/K5-IKKb mice than in Ink4a/ArfKO mice (Fig. 4B; P <0,0001). Representative examples of SCCswith increased levels ofmalignancy, from well-differentiated SCCs to highly undifferen-tiated cutaneous SpSCCs are shown in Fig. 4C–H. SpSCC is a rarevariant of SCC in which tumor cells with spindle-shaped appear-ance infiltrate the dermis diffusely, without formation of epithe-lial nests or cords (34); this tumor type is so malignant andundifferentiated that determination of its origin is sometimesdoubtful as spindle keratinocyte appearance is very similar to thatof malignant cells from fibrosarcomas or melanomas. We havestudied the expression patterns of keratin K5, vimentin, andprotein S100 (epithelial, mesenchymal, and melanocytic cellmarkers, respectively), to ensure the epidermal origin of uncertain

21%

14%

32%

27%

3%3%Ink4a/Arf KO

(n = 63)

Benign tumors Tumors with microcarcinomas Well differentiated SCCs

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4

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12

7 8 9 10 11 12 13 14

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ors/

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se

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019%12%

14%

24%

Ink4a/Arf KO/ K5-IKKb(n = 58)

Moderately differentiated SCCs Undifferentiated SCCs Spindle-cell SCCs

B

K5 Vimentin S100

D

F

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KO

C

G

KJI

H

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5-IK

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Figure 4.

Deletion of the Ink4a/Arf locus abrogates the tumor protective activity of IKKb. A, Tumor multiplicity in K5-IKKb transgenic mice and control littermates in Ink4a/ArfKO background subjected to a two-stage DMBA/TPA carcinogenesis protocol. B, Histopathologic classification of tumors obtained in Ink4a/ArfKO

and Ink4a/ArfKO/K5-IKKb mice 14 weeks after the beginning of the treatment with DMBA and TPA. C–H, Histologic aspect of skin tumors with increased levels ofmalignancy in the indicated genotypes: C and F, Well differentiated SCCs, showing abundant foci of squamous differentiation (horny pearls, arrows);D andG,Moderately differentiated SCCs, with nests and cords of keratinocytes infiltrating the underlying dermis andmuscle; E andH, highly undifferentiated SpSCCsshowing spindle shape keratinocytes infiltrating the dermis in a diffuse pattern. I–K, IHC staining of a SpSCC for keratin K5 (I), vimentin (J), and S100 (K).Scale bars, 100 mm.






tumors containing spindle cells and in some other tumors withclear classification; we analyzed 19 tumors from 9 Ink4a/ArfKO/K5-IKKb mice and 8 tumors from 4 Ink4a/ArfKO mice (Table 2).Typically, SpSCCs coexpress K5 and vimentin (Fig. 4I and J) withthe absence of S100 (Fig. 4K; compare with the staining in apositive control shown in Supplementary Fig. S5A and S5B),which differentiate them from fibrosarcomas (positive for vimen-tin but negative for keratins) and spindlemelanomas, tumors thathave been described in Ink4a/ArfKO mice. The low percentage offibrosarcomas in comparison with the data published in ref. 24could be due to differences in genetic background, latency time, ortumorigenesis treatment (DMBA/UV in ref. 24 and DMBA/TPA inthis work).

It is worth noting that we have been able to determine thefollicular origin of SpSCCs, as K5-positive epidermal follicularcells infiltrate the underlying dermis, showing highly dysplasticspindle shape cells and giving rise to a SpSCC (Supplementary Fig.S5C). We have not found consistent differences in the activity ofStat-3, Erk, or Akt pathways between tumor histologic types orgenotypes (Supplementary Fig. S5D). Spontaneous tumorigene-sis affected slightly more strongly the Ink4a/ArfKO/K5-IKKb thanthe control Ink4a/ArfKO mice, as a higher percentage of micedeveloped tumors (Table 1B). In addition, although the numberof tumors is low, a tendency to develop more malignant skintumors was also observed (as 2 of 6 skin tumors in Ink4a/ArfKO/K5-IKKb were highly malignant SpSCCs, while this tumor typewas not observed in Ink4a/ArfKO mice), reinforcing the dataobtained in two-stage skin carcinogenesis experiments.

Taken together, these results indicate that the antitumoral effectof IKKb is mediated by p16 or p19. Even more, in the absence ofthese tumor suppressors, IKKb is not able to protect against skintumor appearance, but instead it seems to increase tumor malig-nancy, as described for other tissues.

DiscussionIKKb is a fundamental multitarget kinase (5), andmice lacking

IKKb in all their cells die during gestation due to excessive hepaticapoptosis (35, 36); the absence of IKKb in epidermis and stratifiedepithelia results in death at the beginning of postnatal life by aninflammatory skin disease mediated by TNF (37). In humans,however, IKKb deletion seems to be less harmful, as homozygousdeletion of the IKBKB gene is not lethal, at least in some cases, butleads to immunodeficiency (38). In agreement with the multiplefunctions of IKKb, its effect on tumoral transformation of the cellscan be mediated by different pathways, such as NF-kB activation(39); adaptation to metabolic and oxidative stresses, increasingthe ability of cancer cells to survive at low glutamine concentra-tion (40); establishment of stemness properties by Wnt pathwayregulation (41); modulation of T-cell–dependent antitumoralimmune response (42); or inactivation of important cell-cyclecontrollers as p53 (2) and p16 (3) among others. As a result ofthese pleiotropic functions, IKKb promotes cellular transforma-

tion and tumor growth in lung, pancreas, and oral epithelia,among others (16, 17, 19), but it also results in an antitumoraleffect in myeloid cells during melanoma tumorigenesis (18).There are two recent reports that illustrate clearly this context-dependent pro- or antitumoral dual activity of IKKb, as they reportthat IKKb in mesenchymal cells has both tumor-promotor andtumor-suppressor roles in intestinal tumorigenesis, probably as aconsequence of subtle differences in the subpopulations targetedin these experiments (43, 44). Here, we have found a surprisingantitumoral role of epidermal IKKb in nonmelanoma skin cancer.This activity does not seem to be exerted by modifications in theepidermal or dermal inflammatory milieus, nor by a diminutionin the amount of stem cells. As skin-specific p65 knockout leads todownregulation of NF-kB and also protects against tumoraltransformation (8), the effect of IKKb overexpression overskin cancer is probably mediated mainly by NF-kB–independentpathways. When we searched for possible mediators of thisskin tumor–protective function of IKKb, we found that K5-IKKbepidermal keratinocytes expressed high levels of several tumorsuppressor proteins (p53, p16, and p19) that could explain thereduced sensitivity to tumoral transformation of IKKb-overex-pressing skin.

Experimental skin carcinogenesis in animals lacking epidermalp53 indicated that the antitumoral activity of IKKb in skin isindependent of p53; so, probably the increased p53 protein levelfound in K5-IKKb keratinocytes is secondary and not directlycaused by IKKb. Interestingly, the antitumoral effect of IKKb iseven stronger in a p53EKObackground than in ap53wt background(Figs. 1A and 3A); this could be explained by the mutual inhib-itory activity described for IKKb and p53, that would weaken theeffect of IKKb in the presence of p53 (2, 45).

IKKb protection against skin cancer, on the contrary, actsthrough p16, p19, or both. Although some of the activities ofp16 and p19 proteins are different, and even opposite (46), bothof themhave strong andnonredundant tumor suppressor activity.The lack of either p16 or p19 alone results in increased cancersusceptibility, and is less harmful than the combined absence ofboth proteins (47). Favoring the possible leading role of p19 overp16 asmediator of skin IKKb antitumoral activity are the facts thatp19 is expressed at higher level than p16 in skin keratinocytes inqRT-PCR experiments and that mice with one of these proteinsknocked-out confirm the higher general importance of p19 (47).If the main actor in IKKb-mediated tumor protection were p19, itwould exert its antitumoral function by p53-independent func-tions, as p53 absence does not diminish the skin antitumoractivity of IKKb. In any case, the generation of K5-IKKb micelackingp16orp19 individually and the studyof their sensitivity toskin cancer would be required to clarify which of these proteinsmediates IKKb skin-suppressive cancer functions.

It is interesting to highlight that skin overexpressing IKKbgives rise, in the absence of p16 and p19, to SpSCCs, both afterDMBA/TPA treatment and spontaneously (Fig. 4B; Table 1B).Spindle-cell SCC is a rare type of skin tumor, more frequent in

Table 2. IHC classification of uncertain SCCs and fibrosarcomas arisen in Ink4a/ArfKO/K5-IKKb and Ink4a/ArfKO mice treated with DMBA/TPA

Tumor typeGenotype Well-diff. SCCs Undifferentiated SCCs Spindle-cell SCCs Fibrosarcomas Total

Ink4a/ArfKO/K5-IKKb 0 5 14 0 19Ink4a/ArfKO 3 3 1 1 8

NOTE: Classification is based on staining for keratin K5, vimentin, and S100, as described in the text. Some well-differentiated and undifferentiated SCCs wereincluded for control purposes.

Page et al.





immunosuppressed individuals. It has been described, in two-stage chemical skin carcinogenesis, that SpSCC is associated tolower expression level or to deletion of p16 and p19 (28). Ourresults also suggest that this malignant carcinoma is favored byincreased expression of IKKb, as it is found preferentially in Ink4a/ArfKO/K5-IKKb.

The generation in recent years of a growing body of expressionand genomic data from tumoral samples supports the idea thatIKKb is a truly importantmolecule inhuman cancer development.So, the data included in the catalogue of somatic mutations incancer (COSMIC; http://cancer.sanger.ac.uk/cosmic) indicatethat IKKb is frequently overexpressed (sometimes in associationwith copy number gain) in several human cancers, mainly inesophagus (24.8%of tumors), large intestine (18.5%), and breast(9.6%). Interestingly, IKKb is also overexpressed inmore than 7%of the samples of malignant melanomas and of stomach, ovary,and lung cancers; unfortunately, there are not data available forNMSC. Data in The Cancer Genome Atlas (TCGA) indicate thatIKKb is altered (mostly amplified) in around 5%of head and necksquamous carcinomas; in 5%–10% of the cases of ovarian serouscystadenocarcinoma, colorectal and stomach adenocarcinomas,liver hepatocellular adenocarcinoma, uterine corpus endometrialcarcinoma, lung adenocarcinoma, lung SCCs, and esophagealcarcinoma; and over 10% of the cases in breast invasive carcino-ma, and bladder urothelial carcinoma. Interestingly, TCGA dataindicate that IKKb is not altered inNMSC, although the number ofsamples analyzed is low (48, 49). Overall, these data confirm andexpand the growing body of evidence obtained in geneticallymodified animal models, indicating that IKKb overexpressionfavors tumor growth in many cells types, but not in epidermalkeratinocytes, where IKKb prevents tumor development. Thisrepresents a warning note against the proposed treatment ofinflammatory and tumoral diseases with IKKb inhibitors (50),as ubiquitous pharmacologic IKKb inhibition could favor skintumor appearance.

In summary, we have found and described for the first time astrong skin tumor–suppressive function of IKKb. Mechanistically,this antitumoral activity is mediated by p16 or p19, but not byp53. In addition, IKKb seems to cooperate with lack of INK4A/ARF in the genesis of SpSCCs.Overall, these results draw attentionto the need of a careful evaluation of therapies aimed to IKKbinhibition in the treatment of inflammatory and tumoral diseases.

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

DisclaimerThe results shown here are in part based upon data generated by the TCGA

Research Network (http://cancergenome.nih.gov/).

Authors' ContributionsConception and design: A. Page, A. RamirezDevelopment of methodology: A. Page, A. Bravo, C. Lorz, C. Segrelles,J.M. Paramio, A. RamirezAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): A. Page, A. Bravo, C. Suarez-Cabrera, J.P. Alameda,M.L. Casanova, J.C. Segovia, A. RamirezAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): A. Page, A. Bravo, C. Suarez-Cabrera, J.P. Alameda,M.L. Casanova, J.C. Segovia, J.M. Paramio, M. Navarro, A. RamirezWriting, review, and/or revision of the manuscript: A. Page, A. Bravo,J.C. Segovia, J.M. Paramio, M. Navarro, A. RamirezStudy supervision: A. RamirezOther (histopathological studyof the samples, diagnosis and classificationoftumors, and obtaining microphotographs): A. Bravo

AcknowledgmentsThe authors would like to thank Rebeca Sanz, Berta Hernanz, Nerea Guijarro

for their valuable technical help; Rebeca S�anchez-Domínguez and OmairaAlberquilla for their help with the flow cytometry studies; Federico S�anchez-Sierra and Pilar Hern�andez for excellent histologic processing of the samples;and Edilia de Almeida and the personnel of the CIEMAT Animal Unit for micecare and for their help with mice treatments. We also thank Manuel Serrano(Centro Nacional de Investigaciones Oncol�ogicas, CNIO, Spain) for his gen-erous gift of Ink4a/ArfKO mice and Anton Berns (Netherlands Cancer Institute,NKI, the Netherlands) for supplying p53EKO mice.

Grant SupportThis research was supported partially by funds from Fondo Europeo de

Desarrollo Regional (FEDER) and by grants from the Spanish government(PI14/01403, to A. Ramírez; PI16/00161, toM.L. Casanova; SAF2015-66015-R,ISCIII-RETIC RD12/0036/0009, PIE 15/00076, and CB/16/00228, to J.M.Paramio; and RD16/0011/0011, SAF2014-54885-R, and RTC2015-3393-1, toJ.C. Segovia).

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received March 23, 2017; revised May 12, 2017; accepted May 25, 2017;published OnlineFirst June 5, 2017.

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2017;15:1255-1264. Published OnlineFirst June 5, 2017.Mol Cancer Res Angustias Page, Ana Bravo, Cristian Suarez-Cabrera, et al.

DependentInk4a/Arf--Mediated Resistance to Skin Cancer Development Is βIKK

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IKKb-Mediated Resistance to Skin Cancer Development Is ......Oncogenes and Tumor Suppressors...

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